1. Field of the Invention
The invention relates to a novel mutant of tissue factor pathway inhibitor (TFPI) protein and its corresponding DNA sequence. The mutant can be found in humans who show or may show an increased risk of thrombotic diseases. The DNA sequence according to this invention differs from the known TFPI coding DNA by a single nucleotide polymorphism which results in a change of one amino acid position within the known TFPI protein. By screening samples of human blood for said DNA or fragments of it, it is possible to predict a dispostion of thrombotic disorders by which prophylactic application or measures can be initiated.
2. Description of the Related Art
Tissue factor pathway inhibitor (TFPI) is an important regulator in the extrinsic blood coagulation pathway. Although the regulatory biochemical role of TFPI is evident, the clinical significance of this proteinase inhibitor remains to be elucidated. The definition of a clinical TFPI deficiency seems to be more complex than that of other coagulation inhibitors because the activity and concentration of circulating TFPI can not be considered a true measure of in vivo levels. Its determination in plasma samples by immunological methods or functional assays has been shown to be inadequate in the detection of a clinical deficiency.
TFPI is a single chain glycoprotein present in plasma in trace amounts. It was previously known as extrinsic pathway inhibitor, or lipoprotein associated coagulation inhibitor. TFPI belongs to the class of Kunitz-type proteinase inhibitors, and the mature protein contains an acidic amino-terminal end followed by three Kunitz-type inhibitory domains and a basic carboxy-terminal end. The cDNA coding for TFPI was cloned and characterized by Wun et al. (J. Biol. Chem. (1988), 263). The mature molecule consists of 276 amino acid residues, including 18 cysteins (SEQ ID NO: 2), all involved in disulfide bonds, and contains three potential N-linked glycosylation sites. The molecular weight of the polypetide backbone is about 32 kDA; the protein present in plasma runs, however, on SDS-PAGE with an apparent molecular weight of about 42 kDa, presumably due to glycosylation.
The multivalent protease inhibitor is an important regulator of the extrinsic pathway of blood coagulation through its ability to interact with the blood coagulation factor VIIa/tissue factor complex and the activated factor X via its Kunitz-type domains xcex41 and xcex42 (see: Girard, T. J. et al. , Nature 338, 518-520 (1989); Broze, G. J. Jr. et al.; Blood 71, 335 (1984); Rapaport, S. I. and Rao, L. V. M., Thrombosis and Haemostasis 74, 7-17 (1995); Broze, G. J. Jr. , Haemostaseologie 17, 73-77 (1997) ). There is also evidence that infusion of recombinant TFPI may protect against disseminated intravascular coagulation induced by TF or E. coli to protect against venous thrombosis and to prevent rethrombosis after successful thrombolysis in arterial thrombosis. The intravascular distribution of TFPI is complex. The mature human tissue factor pathway inhibitor protein is mainly synthesized by the vascular endothelium (Bajaj, M. S. et al. Proc. Natl. Acad. Sci. USA 88, 8869, (1990)). It has also been detected in at least four intravascular pools: bound to the endothelial cell surface, associated with lipoproteins, carrier-free within the plasma, and sequestered in platelets (Sandset, P. M. and Abildgaard, U., Haemostasis 21, 219 (1991)) A review of the regulation and role of TFPI within the extrinsic pathway system is given by Petersen at al. (Thrombosis Research, 79, 1-47 (1995)).
TFPI plays such an important role in the inhibition of the extrinsic pathway that TFPI deficiencies due to mutations in the TFPI gene should enhance the activity of the prothrombinase complex. This increases the thrombin generation and consequently the risk of venous thrombosis. Such a diminished inhibition of thrombin generation is already well known in inherited coagulation inhibitor defects that predispose to thrombosis including deficiency of antithrombin III, protein C and protein S (Dahlbxc3xa4ck, B. Blood 85, 607-614 (1995) ). The most prevalent inherited abnormality which is known to lead to venous thrombosis is the resistance to activated protein C caused by a single point mutation in the factor V gene (Bertina, R. M. et al., Nature 369, 64-67 (1994)).
The invention relates to a novel mutant of tissue factor pathway inhibitor (TFPI) protein and its corresponding DNA sequence. The mutant can be found in humans who show or may show an increased risk of thrombotic diseases. The DNA sequence according to this invention differs from the known TFPI coding DNA by a single nucleotide polymorphism which results in a change of one amino acid position within the known TFPI protein. It is an object of this invention to screen genomic DNA samples of human normal blood donors and thrombotic patients for alterations in the TFPI gene to assess the influence of a modified TFPI in venous thromboembolic diseases.